PROJECT SUMMARY Lysosomal storage diseases (LSD) are rare inherited metabolic disorders caused by defects in the cellular catabolic system. Mucopolysaccharidosis Type IIIC (MPS IIIC or Sanfilippo disease type C) is one such LSD that is caused by deficiency of the enzyme heparan sulfate acetyl CoA: ?-glucosaminide N-acetyltransferase, (HGSNAT) essential for degradation of heparan sulfate, a repeating carbohydrate generally found attached to proteoglycans. This disease causes accumulation of heparan sulfate and results in progressive and severe neurological deterioration early in life. Most patients become demented and die before adulthood but some survive to the fourth decade with progressive dementia. Currently there is no specific treatment for MPS III and enzyme replacement therapy may not be viable as the recombinant enzyme may have difficulty crossing the blood brain barrier. The disease can however be considered as an excellent candidate for so-called chaperone therapy (where active site specific inhibitors or other small molecules restore some activity of a mutant enzyme) because a threshold activity of approximately 10% of the normal level should be sufficient to prevent storage based on in vitro data. Thus, even such a minor increase in residual enzyme activity as the result of chaperone therapy is likely to have an impact on disease pathology and be beneficial for patients. Recently our collaborator Dr. Pshezhetsky has identified an inhibitor and a chaperone for HGSNAT (AT3784) and demonstrated that it could partially restore the deficient enzyme activity in the cells from MPS IIIC patients, however more potent chaperones need to be identified for a therapy for MPS IIIC. The goal of this STTR is to resolve the tertiary structure of HGSNAT and use this information to direct the synthesis of potent inhibitors/chaperones of HGSNAT. Synthesized compounds will be tested in vitro for their ability to increase the residual HGSNAT activity in cultured cells from MPS IIIC patients. Active compounds that increase enzyme activity by > 10% will be further tested for their ability to reduce storage of heparan sulfate and to stabilize the proper conformation and targeting of the mutant enzyme in cultured patient cells. As a stretch goal we will investigate the ability of the best compound to cross the blood brain barrier. Compounds identified in this preclinical study will provide leads for future phase II in vivo testing and optimization that will be performed in the extension of this project (Phase II) using knock-in mouse models of MPS IIIC generated using the CRISPR Cas9 technology. This proposal leverages the vast X-ray crystallography experience of Professor Geoffrey Chang (University of California San Diego), extensive pharmaceutical medicinal chemistry experience of Dr. Joel Freundlich (Rutgers University), MPSIIIC biology expertise of Dr. Alexey Pshezhetsky (CHU Ste-Justine) and drug discovery knowledge of Sean Ekins (Phoenix Nest, Inc.). Dr. Pshezhetsky?s work on this project will be entirely funded by funds outside this grant including Jonah?s Just Begun or the Canadian Institutes of Health Research. If successful, Phase II will lead to a clinical candidate for studies which will leverage our large global network of clinicians and other scientists as needed.